Method of Treating Parkinson&#39;s Disease and Other Movement Disorders

ABSTRACT

The present invention relates, in general, to movement disorders and, in particular, to a method of treating movement disorders, including Parkinson&#39;s Disease.

This application claims priority from U.S. Provisional Application No.60/960,356, filed Sep. 26, 2007, the entire content of which isincorporated herein by reference.

This invention was made with government support under Grant No.1R21-NSO49534 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

TECHNICAL FIELD

The present invention relates, in general, to movement disorders and, inparticular, to methods of treating movement disorders, includingParkinson's Disease.

BACKGROUND

Parkinson's Disease (PD) is one of the most prevalent of theneurodegenerative disorders. PD results from the death of 60-70% of thedopaminergic neurons located in the substantia nigra pars compacta. Themotor symptoms of the disease are characterized by bradykinesia orakinesia, rigidity, resting tremors, and gait disturbances (Fahn, Ann.N.Y. Acad. Sci. 991:1-14 (2003)). Currently, dopamine replacementtherapy with levodopa is the most widely accepted treatment for PD.Chronic use of the drug, however, leads to long-term complications, suchas on-off states and dyskinesia, for about 60% of the patients.Moreover, a large percentage of these patients become unresponsive tothe drug, rendering such pharmacological therapy ineffective after a fewyears.

The most common alternative to dopamine replacement therapy is thedirect electrical stimulation of deep brain areas, such as the basalganglia and thalamus. This therapy is generically known as Deep BrainStimulation (DBS). In patients with severe PD motor symptoms, chroniccontinuous high frequency electrical stimulation of motor brain regions,such as the subthalamic nucleus, globus pallidus, or thalamus, candecrease tremors, rigidity, and bradykinesia. DBS also permits thereduction of dopaminergic medication, minimizing the long-term sideeffects associated with pharmacotherapy.

Unfortunately, DBS requires a stereotactic, high-precision, intracranialsurgical procedure, and its efficacy depends on the accuracy oftargeting brain nuclei. Additionally, DBS surgery can be associated withserious complications, including intracranial hemorrhage (3.9%) andinfections (1.7%). Adverse events related to the device includeelectrode replacement (4.4%), device dysfunction (3.0%), infection(1.9%) and migration (1.52%) (Kleiner-Fisman et al, Mov. Disord. 21,Suppl 14:5290-5304 (2006)). Thus, despite clear advantages of DBS overL-DOPA therapy, its use is typically restricted to patients in the latestages of PD that are medically stable enough to undergo surgery. Thatrestricts considerably the total number of patients who can benefit fromthis rather invasive neurosurgical procedure.

The precise neural mechanism by which DBS exerts it effects remainsunknown. However, some evidence suggests that its action occurs throughthe disruption of characteristic aberrant low frequency (<10 Hz)synchronized activity of neuronal populations of basal ganglia and/ormotor cortex observed in parkinsonian states (Brown, Mov. Disord.18:357-363 (2003); Brown et al, Exp. Neurol. 188:480-490 (2004); Costaet al, Neuron. 52:359-369 (2006); Gatev et al, Mov. Disord. 21:1566-1577(2006)).

The present invention provides new approaches to the utilization ofneurostimulation as a continuous therapy for controlling the primarymotor symptoms of PD and other movement disorders (e.g. essential tremor(Benabid et al, Lancet 337:403-406 (1991))). The methods to which theinvention relates are less invasive than DBS and avoid the complicationsassociated with intracranial surgery.

SUMMARY OF THE INVENTION

The present invention relates generally to methods of treating movementdisorders, such as PD, as well as other neurological disorders andcertain psychiatric disorders. The methods comprise minimally invasiveor non-invasive stimulation of, for example, peripheral cranial nervesor the spinal cord. The methods result in the disruption of the type ofpathological synchronous activity observed, for example, in the brainsof patients suffering from certain neurodegenerative diseases anddisorders, including PD.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating a non-limiting examplestimulation system.

FIG. 2 is an illustration of a non-limiting example wherein electricalstimulation is delivered to a nerve using a cuff structure.

FIG. 3 is an illustration of a non-limiting example wherein electricalstimulation is delivered to a nerve/neural pathway (e.g., spinal cord)using contact electrodes.

FIG. 4 is a schematic diagram of an embodiment of a system of thepresent invention for effecting electrical stimulation of posteriorfuniculi. (A) The stimulation electrode consists of two parallelplatinum bands (each 2 mm in length, 0.3 mm in width and 0.025 mm thick)separated ˜0.3 mm and embedded in surgical silicone (sylgard). Each bandis connected to a wire (Teflon-coated 7-strand stainless steel, 0.001inches, bare diameter). (B) The electrode is implanted above the dorsalspinal cord and the connections wires are passed subcutaneously to anincision in the head skin and joined to a special connector attached tothe skull. The connector is plugged to 2 stimulus-isolator units, whichprovide biphasic constant-current pulses at desired frequency andintensity. (C) Left: schematic dorsal view of the implanted electrodeand the spinal cord. Right: schematic sagittal view of the implantedelectrode. The electrode is inserted between vertebrae T1 and T2 andlocated longitudinally in close proximity to the dorsal surface of thespinal cord, over the posterior funiculi. Both ends of the electrode aretrapped between the vertebrae and the spinal cord. r. rostral; c:caudal.

FIG. 5 illustrates a single neuron and local field potential activityrecorded simultaneously with locomotive activity in a mouse partiallydepleted of dopamine during bilateral electrical stimulation of theinfraorbital nerve. Top: Experiment protocol, a step in the black lineindicates infraorbital stimulation (IS) at the specified frequency.Black arrow indicates the α-methyl-p-tyrosine (AMPT) injection. Next 7panels are the firing rates assessed in 10-second bins for 3 neurons ofthe dorsolateral striatum (dls-dls3) and 4 neurons of the primary motorcortex (m1-m4). The next 2 panels are spectrograms, representing theoscillatory power of local field potential (a measure of synchronizedactivity of neural populations) as function of time in the striatum(dis) and primary motor cortex (m). Warm colors represent high poweroscillations. Lower panel represents the traveled distance by the mousein 10-second periods.

FIG. 6 shows a partial dopamine depletion produces strongsynchronization at 4 Hz in the corticostriatal circuit. Blue line is theaverage power spectra in normal condition; red line is the average powerspectra after AMPT injection. Data are from 2 mice (rows). Brainstructures indicated on top.

FIG. 7 illustrates effect of infraorbital stimulation on spectral indexand locomotive activity. The spectral index was computed in nonoverlapping 10-second windows and plotted against time (red and greenlines, striatum and motor cortex, respectively). In basal condition, theindex has a value around 0.5, while after AMPT injection (black arrow)it tends to 1. The application of IS at certain frequencies can bringback the index to basal values, for example, at 60 and 120 Hz in Animal1 (upper panel), and at 130 Hz in Animal 2 (lower panel). Concomitantly,locomotive activity (gray line) is increased during these episodes.Although there is a decrease of the spectral index and an increase oflocomotive activity in some of the rest periods, this can be attributedto a residual effect of the immediate previous stimulation.

FIG. 8 illustrates an electrical stimulation of posterior funiculiinduces locomotive activity in dopamine depleted mice. Animals weresubjected to 4-7 cycles of 5 different stimulation paradigms. Eachstimulation epoch lasted 30 seconds. Top row shows the averagelocomotive activity for 7 cycles for a single animal. Bottom row showsthe average results for 5 repetitions for another animal. The paradigmitself is indicated in the top of each plot. The black bar in every plotindicates the stimulation period. The air pulse paradigm was used as acontrol; it consisted of eight air pulses delivered regularly during 30seconds and aimed directly at the animal in order to produce nonspecific stimulation and arousal. Tonic stimulation consists of pulsesat the indicated frequency delivered in a continuous way, while trainindicates the delivery of a train of 20 pulses at 100 Hz every 2seconds. Electrical stimulation at 100 Hz, 100 Hz train, and 300 Hz isextremely effective for inducing locomotion in Animal 1; in Animal 2,stimulation at 10 Hz and 300 Hz induces moderate locomotion.

FIG. 9 illustrates an electrical stimulation of posterior funiculichanges the firing rate of motor cortex and striatal neurons. (A)Average firing rate of example neurons across 7 periods of stimulationat 300 Hz. Black bars indicate stimulation period. Top row shows a motorcortex neuron (left) and a striatal neuron (right) that exhibit adramatic increase in firing rate during stimulation. Bottom row shows amotor cortex neuron (left) and a striatal neuron (right) that decreasetheir firing rates during 300 Hz stimulation. (B) A response matrix for10 M1 neurons and 9 STR neurons recorded in Animal 1. Each rowrepresents the response of a neuron to 6 stimulation paradigms(columns). Red compartments specify a significant increase in firingrate, while blue compartments point to a significant decrease in firingrate (p<0.01, paired T-test).

FIG. 10 illustrates an electrical stimulation of posterior funiculimodifies the temporal activity patterns of neuronal populations in motorcortex and striatum. Average time-frequency charts from 7 recordingsites in M1 and 8 recording sites in STR, across 7 stimulation cycles isshown (first and third columns). The abscissa corresponds to time (blackbar indicates stimulation period), the ordinate axis corresponds tofrequency of oscillatory activity, while the color code indicates theamplitude or power of the oscillations. Warmer colors indicate higherpower at that particular time and frequency. In order to detectsignificant changes in oscillatory power, the data were expressed interms of standard deviations (σ) from the 30 seconds previous tostimulation for every frequency band (standardized power). Thus, highlysignificant increases in power will appear red, while significantdecreases in power will appear blue. While all the stimulus paradigms,including air pulses, induce a decrease in low frequency power (<20 Hz),only electrical stimulation at 100 Hz tonic, 100 Hz train and 300 Hzclearly increases gamma (30-90 Hz) oscillatory power, a prominent neuralfeature correlated to motor activity. The horizontal bands observed inthe 10 Hz paradigm correspond to electrical artifacts of stimulation.

FIG. 11 shows that tissue content analysis confirms that acutepharmacological dopamine depletion in wild-type mice leads to striataldopamine levels similar to those observed in PD patients. Two i.p.injections (250 mg/kg), administered 2 hours apart, of the tyrosinehydroxylase inhibitor alpha-methyl para-tyrosine (AMPT) in wild-typeC57/BL6J mice, reliably decreased striatal levels of dopamine (A) andits metabolites 3,4-Dihydroxy-Phenylacetic Acid (DOPAC) (B) andHomovanillic acid (HVA) (C), as measured 4 hours after the last AMPTinjection (see FIG. 14). Average quantities in depleted animals werereduced for dopamine, DOPAC and HVA to: 4.5 ng, 0.17 ng and 0.41 ng permg tissue, respectively, compared to 14.4 ng, 0.74 ng and 1.36 ng per mgtissue, for saline injected control animals (**=p<0.005, Mann-Whitneytest, n=6 in each group). Tissue content analysis of dopamine andmetabolites.

FIG. 12 illustrates an acute pharmacological dopamine depletion causesparkinsonian like tremor. (A). Examples of 5 min EMG recordings fromnuchal muscles during rest from a depleted animal (red) and a controlanimal (green; rectified voltage was summed in 100 ms time bins). Notethe periodic bursts in the dopamine depleted state in contrast to thealmost atonic state in control conditions. (B). Auto-correlogram of timebins (shown in a) with an amplitude greater than twice the mean. Burststended to occur with a ˜3 s period, although this interval variedslightly over time and between animals. (C). High-frequency componentswithin bursts. Examples of frequency spectrums from nuchal muscles in aWT mouse during two 30 min recordings before (green) and after (red)dopamine depletion; several differences in the distribution of spectralpower is discernable, e.g. the peaks at ˜16.5 Hz and 25 Hz with higherharmonics. Inset shows temporal appearance of the bursts.

FIG. 13 illustrates an acute inhibition of dopamine synthesis produces aparkinsonian state. (A). The average amount of locomotion displayed persecond in the open-field is significantly reduced after acutepharmacological dopamine depletion in wild-type mice (mean and SEMshown, p<0.001, Mann-Whitney). (B). A preferential reduction of fastermovements reflects the bradykinesia in the depleted state; number ofevents expressed as percentage of non-depleted values for the threeintervals are shown. (C). Examples of local field potential (LFP)spectrograms and firing rate plots recorded in MI during two 5-minuteperiods in the same animal, before and after dopamine depletion. Toprow: locomotion during recording periods, second row: LFP power, thirdrow: LFP power standardized to the non-depleted 5-minute period. Notethe increased power in low frequencies in the depleted state (*) and thenormalization of spectral power upon locomotion (red arrow). Bottom row:average firing per second for 6 M1 units. (D). Set-up for electricalstimulation of dorsal columns: The stimulation electrode (red) isimplanted above the dorsal spinal cord and connection wires are passedsubcutaneously to a special connector attached to the skull. Twostimulus-isolator units provide biphasic constant-current pulses atdesired frequency and intensity. (E). Schematic dorsal (left) andsagittal view (right) of the implanted electrode. r: rostral; c: caudal.

FIG. 14 shows a summary of experimental protocol for DCS evaluation inacutely dopamine-depleted wild-type mice. Each animal was stimulatedusing six different types of stimuli (applied every tenth minute) inthree identical cycles during both control conditions (green bars) andin the depleted state (red bars). After baseline data had been acquired,the animals received two AMPT (250 mg/kg) injections, 2 h apart. Whenthe animal displayed clear catalepsy acquisition of data related to thedepleted state was initiated.

FIG. 15 shows s summary of locomotion induced using differentstimulation paradigms. Average locomotion scored per second in responseto the different stimulation paradigms used (30 cycles per paradigm in 9animals). In spite of the akinesia and bradykinesia displayed in thedepleted state, these animals moved almost as far as control animalsduring stimulation periods using dorsal column stimulation. Yellow bardenotes the extent of stimulation period and black line is the meanactivity during a 240 s-period before and after stimulation onset.

FIG. 16 illustrates DCS restores locomotion and desynchronizescorticostriatal activity. (A). Relative change in amount of locomotionin depleted and non-depleted mice (DCS frequencies specified on x-axis,n.V: trigeminal nerve stimulation; mean and SD shown, means for allconditions before and after depletion are significantly different,α=0.005). (B). DCS preferentially increases the fraction of fastmovement components in dopamine depleted animals but not in controls.(C). Average spectrograms of striatal LFPs and firing rates recordedaround twenty-one 300 Hz stimulation events (yellow bar), top row: LFPpower (black trace denotes spectral index, see main text) second row:LFP power standardized to first 240 s. Standardized firing rate to first240 s of 98 striatal and 96 cortical units (row 4 and 5, respectively;firing rate during stimulation period was omitted because of potentialartefacts). Neurons exhibiting significant changes during the 30s-period following stimulation (black line) are indicated with red andblue rectangles (excitatory and inhibitory responses). Middle row:Average locomotion (n=36 events).

FIG. 17 shows a spectral composition and neuronal entrainment tostriatal local field potentials in depleted and non-depleted states.(A). Example of power spectral densities for a single animal (mean andstandard deviation of 375 non-overlapping 4-second periods (in total 25min). Depleted condition (red) showed stronger oscillations around 1.5-4Hz and in the lower beta range (10-15 Hz), whereas the power ofoscillations >25 Hz was decreased in relation to non-depleted condition(green). (B). Spectral power (median±median absolute deviation) ofstriatal LFP oscillations from 9 animals in control and depleted state(25 m-periods). Significant differences were found for all studiedfrequency ranges (***, α=0.001; Mann-Whitney test). (C). Example of astriatal unit entrained to the LFP recorded in the same brain structureduring dopamine-depletion. Top row shows the STA LFP during control anddopamine-depleted states. Bottom row shows the power spectra calculatedfrom the STA (black trace), while dashed red line denotes significancelevel (α=0.001) for the spectral power values. (D). Preferred STA powerof 85 striatal neurons in control (green) and dopamine-depleted (red)state from 9 animals (median±median absolute deviation).

FIG. 18 illustrates activity patterns during spontaneous locomotion.(A). Average spectrogram (window of 1.024 s, slid every 0.5 s) ofstriatal LFP aligned to the onset of spontaneous locomotion in controlcondition (n=1 15 events) and dopamine-depleted condition (n=51 events).The gradual shift from lower to higher frequencies indicated by theaverage spectral index (black trace) starts before locomotion onset(dashed white line). (B). Standardization of spectrogram relative topreceding non-locomotion periods (average spectrogram of 112 stationary10 s-duration events). (C). Firing rate (binned at 0.5 s) of striataland MI units around the onset of spontaneous locomotion. Significantchanges in firing rate (as compared to stationary period) are indicatedwith magenta crosses (excitatory) and dark blue crosses (inhibitoryresponse). (D). Average locomotion during recorded events.

FIG. 19 shows that DCS restores locomotion in severely dopamine-depletedmice and in chronically lesioned rats. (A). The cumulative amount oflocomotion scored in animals receiving DCS in combination withsuccessive L-DOPA injections (black) was significantly higher at alltime points than what was observed for the group only receiving L-DOPA(grey). (B). DCS (yellow shaded area) induced a prominent increase inaverage locomotion per second in 6-OHDA lesioned rats (shaded areaaround trace is SEM). A residual effect of the stimulation can beobserved up to 100 s after stimulation is turned off. The average amountof locomotion for the same rats during normal conditions was obtainedfrom non-DCS sessions. In the sham group, in contrast, the averagelocomotion per second decreased compared to non-DCS sessions (mean±SEM,n=64 stimulation and 64 control sessions for both sham treated andlesioned rats). (C). DCS specifically increases locomotion in 6-OHDAlesioned rats (mean and SEM shown; all means are significantly differentto the others, p<0.001, Kruskal-Wallis and Dunn's multiple comparisontest; flashes indicate DCS sessions). (D). A preferential relativeincrease of fast locomotion episodes was found in the 6-OHDA lesionedgroup reflecting alleviation of bradykinetic symptoms. Ratios of the sumof recorded locomotion episodes in three speed intervals [DCS/non-DCSsessions] are shown (blue: sham, red: 6-OHDA).

FIG. 20 shows local field potentials are shifted to higher frequenciesas a result of DCS even in severely dopamine depleted animals. Averagespectral indices (power ratio: [25-55]/[1.5-25] Hz) calculated forDCS+L-DOPA (black) and L-DOPA (grey) treated animals, respectively. DCSinduced spectral changes even at the lowest L-DOPA dose tested (a single1 mg/kg injection) while the spectral change in the L-DOPA only groupoccurred during the last hours of the testing period (after more than 20mg/kg L-DOPA in total) coinciding with onset of locomotion.

FIG. 21 shows denervation of dopaminergic input to the striatum in6-OHDA lesioned rats confirmed by immunohistochemistry for the enzymetyrosine hydroxylase. (A) coronal sections from a vehicle (0.05%ascorbate saline) injected rat. (B) coronal sections from a rat injectedwith 6-OHDA in three different sites on each side (7 μg per site, 3.5mg/ml). The panels include sections from ˜2 mm anterior to ˜2 mmposterior of Bregma (spacing between consecutive sections presented is80 μm and slice thickness is 40 μm). Quantitative analysis of tyrosinehydroxylase staining in the sections shown indicated a reduction to 21%in 6-OHDA lesioned animals compared to sham treated controls.

FIG. 22 illustrates structures potentially responsible for DCS inducedlocomotion. DCS is thought to primarily activate ascending primaryafferents of the dorsal column pathways terminating in the dorsal columnnuclei. The signals may activate various cortical areas either throughgeneral arousal systems such as the reticular activating system in thebrainstem or by transmission through the leminscal pathway. Boththalamus and cortex exhibit direct excitatory effect on the projectingneurons in the striatum, thereby potentially allowing DCS to modifyactivity patterns in this cell group. Activity in striatal projectionneurons can, in turn, disinhibit motor regions in the brainstem throughinactivation of cells in the output nuclei of the basal ganglia (globuspallidus intema, substantia nigra pars reticulate and ventral pallidum)and thereby enable the generation of motor commands to the spinal cord.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to neurostimulation methods for disruptingthe pathological synchronous activity observed in the brains of patientssuffering from certain movement or motor diseases/disorders, including,but not limited to, PD, cortical or subcortical stroke, amyotrophiclateral sclerosis, essential tremor, Creutzfeldt-Jakob disease, multiplesclerosis, ataxia or other cerebellar disorder, as well as dystonia ordyskinesia. The present methods can also be used in the treatment ofpatients suffering from other neurological disorders or from certainpsychiatric disorders (e.g., patients suffering from various types ofepilepsy, depression or obsessive compulsive disorder). In contrast toDBS, these methods involve either minimally invasive or non-invasivestimulation (e.g., electrical stimulation) of peripheral cranial nerves,such as the trigeminal and vagus nerves (advantageously, uni- orbilateral stimulation of the maxillary branch of the trigeminal nerve(or the vagal nerve unilaterally)), the dorsal sensory roots of spinalnerves, or the posterior funiculus of the spinal cord that form theascending fibers of the medial lemniscus-dorsal column pathway of thesomatosensory system. The advantages of using peripheral nerves (or thespinal cord) as the focus of the stimulation protocol are significant.In addition to requiring minimal or no surgical intervention to apply anelectrode to these nerves and neural pathways, this approach also hasthe advantage that many more stimulation protocols can be tested in eachpatient in order to determine the optimum protocol. Further, reducedpower requirements suitable for use in these approaches eliminate theneed for subcutaneous implantation of large batteries. In addition, thisparadigm reduces to zero the risk of brain hemorrhage (the main and mostserious side effect of DBS), greatly minimizes or totally eliminates therisk of brain infection, and reduces significantly the likelihood ofrejection of chronically implanted foreign materials. These advantagesincrease many fold the total number of patients who can benefit fromneurostimulation therapy alone or in combination with other therapeuticstrategies (e.g., dopamine replacement in the case of PD patients),while significantly reducing the associated morbidity.

In accordance with the present invention, chronically implantedperipheral nerve cuff electrodes, stimulators of the posterior funiculi,or electrical stimulators applied to the skin surface are employed togenerate an essentially innocuous electrical stimulation that disruptssynchronous neural activity in higher brain centers, such as the motorcortex and the basal ganglia. Disruption of this synchronous neuralactivity, which precludes initiation of voluntary movements (akinesia),provides continuous relief of the symptoms experienced by patients(human or non-human animal patients) with movement diseases/disorders,for example PD, or other neurological disorders or certain psychiatricdisorders.

FIG. 1 illustrates a non-limiting example stimulation system 10. Astimulator 12 generates a stimulating signal that is delivered to atarget area 20 of a patient through suitable conductors 18 and contactpoints 22. The area 20 corresponds to or is proximate to one or moremotor-related nerves or neural pathways. Non-limiting examples of suchareas include infraorbital nerves, posterior funiculi of the spinalcord, and epidurally above the nerve or neural pathway to be stimulated.

The stimulator 12 can generate an electrical, optical, or any othersuitable type of stimulating signal. In a preferred, non-limitingimplementation, the stimulator 12 generates and delivers an electricalstimulation signal. In that case, the conductors 18 are electricalconductors and the contact points 22 are electrically conductive contactpoints. Preferably, the stimulator 12 is a small electronic circuit thatcan be implanted under the skin of, or disposed outside the body of, thepatient.

Non-limiting examples of commercially available electrical stimulatorssuitable for use in the invention include the electrical MuscleStimulator EMS 400 available from ReliaMed, Brooklyn Park, Minn. Thestimulator 12 can be powered by an integrated power supply or by aseparate power supply 16, the latter being illustrated with dashedlines. One example power supply is a battery and small sized batteriessuch as lithium batteries may be desirable for human applications.Although not required, it is preferred that the stimulator 12 beprogrammable in order to set or vary (or later adjust) one or morevarious stimulation parameters such as, for example, signal amplitude,frequency, pulse width, pulse asymmetry, polarity, etc.

The objective of the stimulator 12 is to deliver a signal that disruptsaberrant low frequency synchronization in motor related neural circuitsto facilitate volitional movement. In this case, synchronization meansthe coordinated activity of the neural circuit at a low frequency, e.g.,less than 10 Hz. The stimulator 12 disrupts that low frequencysynchronization and consequently the overall activity of the neuralcircuits resembles normal activity patterns which are characterized by ahigher frequency range, e.g. over 30 Hz. Examples of an electricalstimulating signal include electrical pulses emitted at a frequency of100 and 300 Hz applied continuously or in short periods (trains). Pulsedurations can vary. Excellent results have been obtained using biphasicpulses having pulse durations between 250 μsec to 1 msec. Biphasicpulses can vary between themselves in amplitude and duration. The rangeof amplitude or intensity of the stimulating signal depends on thepatient and the effect sought.

There are different ways that the stimulating energy can be delivered tothe area to be simulated. FIG. 2 illustrates a non-limiting examplewherein electrical stimulation is delivered to a nerve using a cuffstructure 30. The cuff can be place around a nerve, for example, andsecured using a tie material 32 which can be silk or other suitablematerial. Other securing mechanism, such as adhesive, clamp, etc., canbe used. The cuff includes two separated conductive bands 34 and 36,each coupled to one of the conductors 18. The bands can, for example, bemade of platinum but other suitable conductive materials can be used.The conductors 18 that connect the bands to the stimulator 12 can beinsulated with suitable material. The two bands 34 and 36 are spaced topermit current flow and to deliver an optimal current density profilefor stimulating the area 20.

Another non-limiting example is shown in FIG. 3 wherein electricalstimulation is delivered to the area 20 (e.g., the spinal cord of apatient) using contact electrodes 40 and 42. In this example, thecontact electrodes 40 and 42 are spaced apart rectangles but they can beother shapes of suitable size to deliver the desired stimulation to area20. As in the example in FIG. 3, the contact electrodes 40 and 42 can bemade of platinum and connected to the stimulator 12 via insulated wireconductors.

In one embodiment of the invention, a stimulatory system as describedabove is used in stimulating an infraorbital nerve. In accordance withthis embodiment, a nerve contact electrode, which is preferably a nervecuff electrode, is in contact with the nerve to be stimulated. Theelectrode is manufactured of a conductive material so as to transmit anelectrical pulse. Additionally, the electrode is preferably treated tominimize any potential physiological reaction to the electrode, and toinsulate the portion or portions of the electrode that do not contactthe nerve. Suitable insulation materials include, but are not limitedto, TEFLON® for a lead wire and SYLGARD® (available from Dow CorningCorp. of Midland, Mich.) for a nerve contact electrode.

As indicated above, the nerve contact electrode is preferably a nervecuff electrode. A nerve cuff electrode is an electrode designed toencircle the nerve to be stimulated, thereby increasing the area ofcontact and stimulation. A suitable nerve cuff electrode can compriseone or more conductive bands optionally mounted on a support surface.Preferably, a conductive band comprises platinum. When a plurality ofconductive bands are employed in a nerve cuff electrode, it ispreferable that the bands be communicatively associated with one anothersuch that when a stimulation pulse is applied to the nerve cuffelectrode it is dispersed through all bands of the electrode. Additionalwire or other material can be affixed to the nerve cuff electrode inorder to permit its emplacement around the nerve to be stimulated. Areasof the electrode through which it is not desired to transmit current canbe coated with an insulator.

Leads from each band of the nerve contact electrode are attached to thepower source such that current passes from one band to the next. Thenerve is activated when current passes from one band to the next.

Nerve cuff electrodes can be implanted by surgically exposing the nerveand orienting the electrodes such that they surround the nerve. Nervecuff electrodes can be implanted either on only one branch of a nerve,(e.g., the left branch of the infraorbital nerve), or on both branchesof the nerve (e.g., the right and left branches of the infraorbitalnerve). Implantation of a nerve cuff electrode on a single branch of anerve present in a subject can facilitate unilateral stimulation of thatnerve. However, implantation of a nerve cuff electrode on two or morebranches of a nerve present in a subject can facilitate bilateralstimulation of that nerve.

As regards stimulation parameters, values will be limited by thetechnical specifications of the stimulator:

Electrode polarity: electrode polarity (when one of the two electrodesis positive or negative) is not necessarily relevant for the achievementof beneficial effects. In the case of trigeminal (infraorbital orsupraorbital) stimulation, bilateral implantation is preferred, since itis more effective than unilateral implantation (Fanselow et al, J.Neurosci. 20:8160-8168 (2000)). In the case of PD it can be important touse bilateral stimulation to conserve the symmetry of the stimulationand produce a comparable effect in both hemispheres of the brain.

Pulse duration: exemplary values are 250 μs, 500 μs and 1 ms. The longerthe pulse duration, the less the current intensity needed to achievedesired effect.

Frequency: for example, 100 Hz up to 300 Hz.

Current intensity: this is a critical parameter to be controlled.Intensity is adjusted for every frequency and pulse durationconfiguration, advantageously, starting with the minimum intensityavailable with the stimulator used. The intensity reported ascomfortable for trigeminal nerve transcutaneous stimulation in humans is<20 mA (DeGiorgio et al, Epilepsia 47:1213-1215 (2006)).

The optimization of these parameters can be carried out in a mannersimilar to the programming of DBS in PD patients (Volkmann et al, Mov.Disord. 21 Suppl 14:S284-289 (2006)), which can be described briefly asfollows. Predetermined values for electrode polarity, pulse duration andfrequency are selected, while intensity is set at the minimum possible.The stimulation is turned on and evaluation of beneficial effect andside effects are performed. This step is repeated while increasingcurrent intensity in small increments. Rigidity is a key aspect forevaluating beneficial effects, since, in DBS, it responds usually withinseconds. Alleviation of bradykinesia and tremor may exhibit variabledelay. Motor tasks described in items 23-29 of the Unified Parkinson'sdisease Rating Scale can also be used. Side effects include theperception of tingling, pain and ultimately, muscle contraction. Thelast two of these side effects are preferably avoided.

The same general strategy can be used to stimulate the dorsal roots orthe sensory branches of spinal nerves in order to obtain a similar motoreffect. FIG. 3 is a schematic of a further embodiment of the invention,that is, a system for effecting electrical stimulation of posteriorfuniculi (see also FIG. 4). Direct electrical stimulation of the dorsalcolumns of the posterior funiculus of the spinal cord, the regioncontaining the ascending fibers of the dorsal-column medial lemniscuspathway, can be used to generate the same motor effects in patients(e.g., PD patients), that is, increase in locomotion activity and blockof peripheral tremor. Posterior funiculi, also known as dorsal columns,convey tactile and proprioceptive input to supraspinal structures. Asshown in Example 2, electrical stimulation of the posterior funiculi canalleviate akinesia in a pharmacological PD model. Consistently,stimulation of the posterior funiculi is also associated withelectrophysiological activity patterns similar to those displayed duringnormal motor activity (see Example 3 below).

Preferred parameters for effecting electrical stimulation of theposterior funiculi are as follows. Electrical stimulation can consist ofbiphasic squared constant current pulses of 2 ms duration delivered atdifferent frequencies. Four stimulation paradigms have been tested inanimals: 10 Hz, 100 Hz, 300 Hz and 0.5 Hz trains each consisting of 20pulses at 100 Hz. In animal studies, the current intensity can beadjusted based on the behavioral response of the animal. Thresholdintensity for each paradigm can be established as the minimum current atwhich a clear associated behavior, such as transient freezing, arousal,or uneasiness, is displayed. For the study described in the Example 2that follows, 1.2-1.5 fold the threshold was used, which corresponds to180-450 microamperes.

Posterior funiculi stimulation (PFS). PFS can be achieved by chronicimplantation of, for example, custom made platinum flat-electrodes (FIG.4A) positioned epidurally above the posterior funiculi of the spinalcord (FIGS. 4B and 4C) at the upper thoracic level. Bilateralstimulation of the cord is preferred.

In addition to the techniques described above for trigeminal and spinalcord stimulation, which are invasive, other non-invasive approaches canbe used to stimulate the trigeminal nerve. For example, transcutaneoustrigeminal nerve electrical stimulation can be used. This method isnon-invasive and fully reversible, allowing for testing effectivenessand tolerability. Commercially available surface electrical stimulatorscan be employed in the practice of this invention. A surface stimulatorcan be positioned on the surface of the face, for example, on top of thetrigeminal nerve. Electrical current delivered by a skin surfacestimulator can be expected to produce the same type of effect observedwith nerve cuff electrodes. For example, electrical muscle stimulatorEMS 400 (ReliaMed, Brooklyn Park, Minn.) can be employed which providesbiphasic asymmetrical pulses of 250 μs duration at a maximum of 120 Hz.The intensity range is 0-100 mA. Dimensions are 27×62×95 mm, and weightis 140 g including the batteries (seewww.vitalityweb.com/backstore/ems.htm). Silver-gel auto-adhesivestimulation electrodes can also be used (Superior Silver-PermagelElectrodes, Tyco Healthcare/Uni-Patch, Wabasha, Minn., U.S.A.), and a 9V DC battery. In accordance with this embodiment, all components,including the electrodes, can be disposed outside the body of thepatient. The auto-adhesive electrodes can be attached to the skin whilethe EMS unit can be carried in a belt or bag.

Percutaneous nerve stimulation electrodes can also be used to effectnon-invasive stimulation. In accordance with this approach, part or allof the stimulation setup is implanted in the patient's body. Self-sizingcuff electrodes suitable for trigeminal stimulation (infraorbital nerveor supraorbital nerve) can be used that are commercially available fromNeurotech (Louvain-la-Neuve, Belgium). A wide variety of quadripolar oroctapolar percutaneous electrodes for posterior funiculi stimulation areavailable from Medtronic, Inc. (Minneapolis, Minn.). As regards theneurostimulator (electrical device generating and delivering theelectrical pulses), fully implantable systems (i.e., wherein theelectrodes and neurostimulator, along with the power source, areimplanted) can be used as can systems wherein only the leads and aradio-frequency receiver are implanted (the power source being wornexternally with an antenna over the receiver). A detailed description ofcommercially available implantable surface electrodes andneurostimulators is provided atwww.medtronic.com/neuro/paintherapies/pain_treatment_ladder/neurostimulation/stimulators_stim_sel/neuro_stim_stim_sel.html.

Certain aspects of the invention are be described in greater detail inthe non-limiting Examples that follows. (See also U.S. Published Appln.20030083716.)

Example 1

It has been possible to disrupt aberrant synchronic activity of neuralpopulations observed in normal mice partially depleted of dopamine ortransgenic mice completely depleted of dopamine using stimulation ofperipheral nerves, such as the trigeminal nerve. In these studies, acuff electrode was chronically implanted around the infraorbital nerve,a branch of the trigeminal nerve. This approach was employed to test theefficacy of peripheral nerve stimulation, as compared to DBS, inalleviating motor impairment in: i) wild type mice in which dopamine waspartially depleted, and ii) a transgenic animal model of PD (the DAT-KOmouse) in which dopamine was almost completed depleted from the brain.Preliminary results show that aberrant low frequency oscillationspatterns of neural activity from the corticostriatal circuit induced bypartial dopamine depletion in wild type mice can be decreased andlocomotive activity restored by infraorbital stimulation (IS).

The first step of these studies consisted of determining the stimulationparameters (frequency and amplitude) optimum for reducinghypodopaminergic related aberrant synchronization in the corticostriatalcircuit. This was achieved by systematic stimulation of the infraorbitalnerve at different parameters along with recording of corticostriatalactivity. Once the optimal values of frequency and amplitude weredetermined, an assessment was made of the effectiveness of thesestimulation parameters in recovering gross locomotive activity andalleviating tremors and rigidity associated with dopamine depletedstates. As control, the IS was compared to direct cortical stimulation,which is known to have effects similar to those to DBS. Theseexperiments made it possible to compare the efficacy of the presentprocedure versus more conventional methods of brain stimulation.

EXPERIMENTAL DETAILS

Parkinson's Disease Model.

Dopamine transporter-knockout (DAT-KO) mice lack the gene encoding thedopamine transporter, which is responsible for the re-uptake ofextra-synaptic dopamine and replenishment of dopamine stores in thepresynaptic terminal (Gainetdinov and Caron, Annu. Rev. Pharmacol.Toxicol. 43:261-284 (2003)). Treatment with the tyrosine hydroxylaseinhibitor, α-methyl-p-tyrosine (AMPT), in these animals preventsdopamine synthesis which, along with the lack of the dopaminetransporter, causes striatal dopamine concentrations to fall to 0.2% ofthe level observed in control animals (Sotnikova et al, PLoS Biol.3:e271 (2005)). As a consequence, DAT-KO mice treated with AMPT displaysevere akinesia, rigidity, and tremors, resembling the terminal stagesof PD in humans, for up to 16 hours (Sotnikova et al, PLoS Biol. 3:e271(2005)). Therefore, dopamine depleted DAT-KO (DDD) mice provide anexceptional model for assessing the effectiveness of peripheral brainelectrical stimulation as a potential therapeutic agent for PD.

Infraorbital Nerve Stimulation.

In mice, IS is achieved by chronic bilaterial implantation of custommade platinum cuff-electrodes in the infraorbital nerve (Fanselow et al,J. Neurosci. 20:8160-8168 (2000)). Square biphasic pulses of 40 μs ofduration, delivered at 1-130 Hz with amplitude of 0.011-0.5 mA, will betested.

Peripheral nerve electrodes consist of two bands of platinum (0.5 mmwide and 0.025 mm thick, ˜0.8 mm separation between bands) that runcircumferentially around the nerve. The platinum bands are held in placeand electrically insulated by a thin Sylgard coating. Each band isconnected to a piece of flexible, 3-stranded Teflon-coated wire that isused to pass current between the two bands. The chronic implantation ofthese nerve cuffs requires general anesthesia in the rodents. Afterdissection of the trigeminal nerve, the cuff electrode is positionedaround the nerve, such that the nerve stays surrounded by the cuff. Thecuff is then tied around the nerve to hold it in place and the surgicalwound sutured. The Teflon-coated leads from the platinum bands runsubcutaneously where they were attached to a battery pack.

Neural Activity Recordings.

In mice, a 32-electrode array is implanted targeting two structuresaffected in PD: dorsolateral (sensorimotor) striatum and primary motorcortex. Activity of single units (spikes) as well as activity ofpopulation or neurons (local field potential, LFP) will be acquired andrecorded by an IMAP system.

Electromyogram (EMG).

Muscular activity will be recorded from a wire attached to trapeziummuscle.

Motor Performance.

The improvement of motor impairment upon IS is assessed by a variety ofstandard procedures described elsewhere as open field activitymeasurement, rotating rod test, akinesia test, catalepsy test, graspingtest of muscular rigidity, bracing test and vertical pole test.

Results

Two C57/J (wild type) male mice where implanted with peripheral nervecuff electrodes, microwire electrodes arrays in the striatum and motorcortex and one EMG wire in the trapezium muscle. After a week ofrecovery, a series of experiments were carried out. Individual animalswere introduced to the open field activity (OFA) monitor cage, whichpermitted tracking of the animal's locomotive activity. After 10minutes, the animal was injected with AMPT (250 mg/kg, i.p.) to producea partial depletion in dopamine brain levels. The stimulation started 15minutes after the injection. It was delivered in 2-minute windows,alternated with 2 minutes of rest (no stimulation). The amplitude wasfixed at 0.4 mA. This value was selected on the basis that it produced anoticeable effect on the animal (soft scratching of the whiskers pads)at the onset of stimulation but no further signs of discomfort or pain.Sixteen different frequency values delivered in a pseudorandom way(1-130 Hz) were tested.

As seen in FIG. 5, dopamine depletion affected the activity of singleunits (see units dis1, dis2, m3, m4). Nevertheless, a dramatic effect onthe population activity can be observed in the spectrogram. Before theAMPT injection, the oscillatory power of Local Field Potentials (LFPs)was broadly distributed between 0-8 Hz, while dopamine depletionproduced a strong synchronization at 4 Hz, revealed as a clear red bandthat appears after time=600 s.

The key observation in these experiments was that IS at some frequenciescould revert this strong neural oscillation pattern to the type ofdesynchronized firing activity observed during the control period. Thiseffect allowed the animal to recover some locomotive activity as seen inthe time period between 3600 and 4800. In order to quantify thisobservation, the ratio between the LFP power in the 2-4 Hz and 0-4 Hzrange was used as an indicator of the predominant oscillatory pattern(FIG. 6). This measure was denominated dopamine spectral index. Adopamine spectral index close to 1 indicates a hypodopaminergicoscillatory pattern, while a spectral index close to 0.5 indicates anormal pattern.

As shown in FIG. 7, the dopamine spectral index clearly distinguishedthe oscillatory pattern observed during the control dopamine depleted(DD) conditions. In order to assess statistical significance, spectralindex values were computed in 10-second windows, and grouped accordingthe stimulation frequency or experimental condition (control ordopamine-depleted (DD)) for a multiple group comparison. In Animal 1, itwas found that the spectral index during infraorbital stimulation at 40,60, 80, 100, 120 and 130 Hz was significantly lower than in DD condition(p<0.01, ANOVA) and not different from basal condition. Of those, onlystimulation at 120 HZ showed a significant recovery of locomotiveactivity (p<0.01, ANOVA). In Animal 2, stimulation at 1, 10, 30, 40, 70and 120 Hz also showed significant differences with DD condition.

The preliminary results in wild type mice showed that the aberrantsynchronized neural activity and the loss of motor activity, bothrelated to partial hypodopaminergic condition (similar to early stagesof PD), could be recovered by electrical infraorbital stimulation. Theseexperiments will be repeated using a PD animal model, such as thedopamine-depleted DAT-KO mouse, to demonstrate that the stimulation ofperipheral nerves is an effective treatment for almost complete dopaminedepletion.

Example 2 Experimental Details

Parkinson's Disease Model.

C57BL/6 wild type male adult mice were used. Animals were treated with250 mg/kg of the tyrosine hydroxylase inhibitor, α-methyl-p-tyrosine,every 2 hours, until akinesia and catalepsy were displayed. The key ofthis manipulation is a decrease of dopamine in the central nervoussystem to less than 40% of normal levels, thus resembling parkinsoniancondition. Akinesia and was assessed by visual inspection, whilecatalepsy was assessed by a custom test. Treated animals exhibited thosesymptoms for several hours after last injection.

Posterior Funiculi Stimulation.

(FIG. 4A) The electrode used to effect dorsal funiculi stimulationconsists of two parallel platinum bands (each 2 mm in length, 0.3 mm inwidth and 0.025 mm thick) separated ˜0.3 mm and embedded in surgicalsilicone (Sylgard). Each band is connected to a wire (Teflon-coated7-strand stainless steel, 0.001 inches, bare diameter). (FIG. 4B) Theelectrode is implanted above the dorsal spinal cord and the connectionswires are passed subcutaneously to an incision in the head skin andjoined to a special connector attached to the skull. The connector isplugged to 2 stimulus-isolator units, which provide biphasicconstant-current pulses at desired frequency and intensity. (FIG. 4C)Left: schematic dorsal view of the implanted electrode and the spinalcord. Right: schematic sagittal view of the implanted electrode. Theelectrode is inserted between vertebrae T1 and T2 and locatedlongitudinally in close proximity to the dorsal surface of the spinalcord, over the posterior funiculi. Both ends of the electrode aretrapped between the vertebrae and the spinal cord. r: rostral; c: cauda

Stimulation Experiment.

Once in akinesic state, animals were subjected to a number ofstimulation cycles. Each cycle comprised 5 stimulation paradigms of 30second duration, regularly distributed in 1 hour. During the cycles,locomotive activity of the animal was measured by means of an array ofinfrared beams displayed in the bottom of the cage. Electrophysiologicalactivity from primary motor cortex (M1) and dorsolateral striatum (STR)was recorded. These structures are related to initiation of volitionalmovement and their activity is severely altered in PD.

Results

Results are shown for two animals. Once in akinesic state, animals donot exhibit locomotive behavior. The delivery of strong air pulses tothe body of the animal, which in a normal situation results in the mousefleeing to another location in the cage, has no such effect in theakinesic animal (FIG. 8). However, stimulation of posterior funiculi atdifferent frequencies will induce moderate to high levels of locomotiveactivity (FIG. 8). Stimulation at 300 Hz appears to be effective in alltreated animals (data not shown). The observed behavior exhibited duringstimulation by different animals is consistent. Once stimulation starts,the animal will stretch all four limbs in a rapid single movement. Aftera few seconds, the mouse will start moving around the field.

In order to identify a possible mechanism for akinesia relieved by PFS,the activity of M1 and STR neurons was analyzed. M1 and STR neuronschange their firing rates (increase or decrease) when the animal isengaged in motor activity (Costa et al, Neuron 52:359-369 (2006)). Sucha trend was observed during PFS stimulation, with neurons increasing ordecreasing firing rate. An example is shown in FIG. 9A. A summary forthe results in Animal 1 is shown in FIG. 9B. PFS at 300 Hz, the mostsuccessful paradigm for inducing locomotion, is also the one thatproduced significant changes in firing rate in most neurons, namely 9out of 10 M1 neurons and 3 out of 9 STR neurons. On the other hand, 10Hz PFS, which is not effective in inducing locomotion in this animal,produce firing rate changes only in 2 out of 10 M1 neurons and 3 out of9 STR neurons.

Local field potential (LFP), which is a measure of synchronized neuralpopulation activity, was also recorded from M1 and STR during PFSdelivery. Dopamine depletion related akinesia, such in PD, is related tostrong synchronization at low frequencies (<10 Hz), which is observed asan increase in LFP oscillatory activity at low frequencies and adecrease in oscillatory power at high frequencies (>30 Hz). PFS at 100Hz and 300 Hz reverse this trend in both M1 and STR, increasingoscillatory power in the range 30-90 Hz and decreasing it at lowfrequencies (FIG. 10). This effect prevails even after stimulationperiod is over (FIG. 10).

In summary, the results indicate that high frequency PFS (100 Hz and 300Hz) is effective in disrupting aberrant low frequency synchronization inmotor related neural circuits, thus facilitating the initiation andexecution of volitional movement.

Example 3 Experimental Details Animals

In total 28 wild-type and 8 DAT-KO C57/BL6J mice and 8 Long-Evans ratswere used. Animals were kept on a 24 hour day-night cycle and receivedfood and water ad libitum. All the procedures involving animals weredone according the protocols approved at Duke University.

Surgery

In a single surgery both stimulation and recording electrodes wereimplanted under deep Ketamine/Xylozine anaesthesia (50-90 mg/kg). Theflat stimulation electrode was inserted between vertebrae T1 and T2 inmice and between T2 and T3 in rats placed longitudinally in the epiduralspace over the dorsal columns, thereby being in close proximity to thedorsal surface of the spinal cord. Both ends of the electrode wereinserted under the laminae of the vertebral arcs of the adjacentvertebrae effectively stabilizing the implant. For trigeminalstimulation, cuff-electrodes were implanted bilaterally in theinfraorbital nerve as previously described (Fanselow et al, J. Neurosci.20:8160 (2000)).

Tissue Dopamine Content Analysis

Striatal tissue content of dopamine was assessed using high performanceliquid chromatography with electrochemical detection, as previouslydescribed. Striata were rapidly dissected, frozen, and stored at −80° C.Later, tissue samples were homogenized in 0.1M HClO4 containing 100ng/ml 3,4-dihydroxybenzylamine (DHBA) as an internal standard.Homogenates were centrifuged for 10 min at 10,000×g. Supernatants werefiltered through 0.22 μm filter and analyzed for levels of dopamineusing HPLC-EC. Monoamines and metabolites were separated on a microborereverse-phase column (C-18, 5 μm, 1×150 mm, Unijet, BAS). The volume ofinjection was 5 μl.

Striatal 6-OHDA Lesions

Rats received a total of 21 μg 6-OHDA, injected in 3 locations of thestriatum on each side. The injections contained a saline solution with3.5 mg/ml 6-OHDA and 0.05% ascorbate. Anteroposterior, mediolateral anddorsoventral coordinates for the injections were: +1.0, +/−3.0, −5.0;−0.1, +/−31, −5.0 and −1.2, +/−4.5 and −5.0 (Winkler et al, Neurobiol.Dis. 10:165 (2002)).

Tyrosine-Hydroxylase (TH) Staining and Quantification

The extent and position of striatal lesions was confirmed by THimmunohistochemistry. For quantification of TH-staining, digital photosof the individual sections were taken with a 16× microscope (Stemi2000-c, Zeiss) under identical illumination conditions. The range inbrightness was practically identical for every photo ranging from nolight (inside the microscope) at the outer edge to the brightestnon-stained part of each section. Images were converted to grey-scalebitmap format with an 8-bit dynamic range in brightness. Pixels with anintensity ranging from ⅕ to ⅔ of maximum were found to include allstained parts of the sections, as confirmed by visual inspection. Thestaining and number of pixels with a brightness corresponding to thisinterval was quantified for all sections taken from the same part of thebrain in a sham treated and lesioned rat, respectively (FIG. 21) and the6-OHDA lesioned animal was found to have only 21% of the sham treatedanimal.

Stimulation Electrodes

The flat electrodes used for dorsal column stimulation consisted of 2platinum bands, for mice: 2 by 0.3 mm and rats: 4 by 0.9 mm (platinumfoil thickness 25 μm; Goodfellow Cambridge Ltd, Huntingdon, England)positioned parallel to each other. Each band was connected to a thinstainless steel wire (teflon-coated 7-strand, 25 μm, bare diameter) withsilver paint at the upper surface. The assembled components wereembedded in surgical silicon, leaving the lower surface exposed. Fortrigeminal nerve stimulation cuff-electrodes surrounding theinfraorbital nerve branch were manufactured according to previouslydescribed techniques, except size was reduced to fit the mice (Fanselowet al, J. Neurosci. 20:8160 (2000)).

Recording Electrodes

S-isonel-coated tungsten wire electrodes (33 μm, California Fine Wire,Calif.) were assembled into 2×8 arrays and cut into two groups with twodifferent lengths matching the distances to the dorsolateral striatumand the infra-granular layer of the primary motor cortex (Costa et al,Neuron. 52:359 (2006)). For EMG recordings, a single-ended stablohmmicrowire electrode (50 μm) was inserted in the nuchal muscles. Themicrowires were attached to a printed circuit board with a miniatureconnector (Omnetics Connector Corp., Minn.) attached to the oppositeside of the circuit board.

Open-Field Experiments

Rats were placed in a circular open-field (90 cm diameter) for 70 min(10 min adaptation and 1 h behavioural recording). The animals were thenallowed to rest in its home cage for one hour. After that, the animalswere reintroduced into the open-field and dorsal column stimulation(DCS) was applied every 10 minutes for 30 seconds. In mice, locomotionwas documented both by video recordings and through an automatedtracking system based in infra-red beams (Med Associates Inc., Vt.).Recordings were obtained before and after dopamine-depletion, and DCSwas applied as described below.

The movements of the rats in the open-field were extracted from thedigitized video recordings, with approximately 1 mm spatial resolution,with custom designed algorithms implemented in command interpretingsoftware (The MathWorks Inc., Mass.). For mice, scored locomotion fromthe infra-red tracking system was binned into 500 ms bins (originalresolution 50 ms). For locomotion extraction from video data, singleframes with a 500 ms interval were used. Locomotion data collected byboth techniques were fully comparable as confirmed by multiplecalibration sessions.

Electrical Stimulation

DCS and infraorbital nerve stimulation consisted of biphasic squareconstant current pulses of 1 ms duration each phase, delivered at 10,100 and 300 Hz (Master-8 Stimulator, A.M.P.I., Israel).

Signal Acquisition

LFPs, EMGs and single- and multi-unit activity were recorded using amultichannel recording system (Plexon Inc, Tex.). LFPs and EMG werepre-amplified (500×), filtered (0.7-170 Hz), and digitized at 1000 Hzusing a digital acquisition card (National Instruments, Austin, Tex.).Action potentials were split-off in the pre-amplifier by a band-passfilter (154 Hz-8.8 kHz) and digitized at 40 kHz.

Data Analysis

LFPs were corrected off-line for frequency dependent phase-shiftsinduced by the hardware filters (Nelson et al, J. Neurosci. Methods169:141 (2008)). To avoid high-amplitude movement artefacts we discardeddata from <1.5 Hz oscillations. Response latencies of LFP changes in MIand striatum following DCS onset was estimated from single pulse evokedpotentials (1.3 T at 0.5 Hz)

Single- and multi-units were sorted according to their wave forms usingthe off-line sorter software from Plexon (Plexon, Inc., Texas). Thecriteria used for single unit classification was that spike shapes wereclearly different from other spikes as judged by a cluster separation infeature space (Lin et al, J. Neurophysiol. 96:3209 (2006)) and theabsence of spiking activity during the refractory period (less than 0.1%of spikes occurring within 2 ms of another spike). Units not fulfillingthe latter criterion were classified as multi-units.

To determine whether a neuron changed its firing rate after a particulartreatment, a firing-rate distribution of the period preceding thetreatment (0.5 s bin for pre-locomotion analysis, 1 s bins for post-DCSanalysis) was generated and the 99% confidence interval calculated basedon a Poisson distribution. A neuron was considered to have changedfiring rate if the firing frequency of at least one (pre-locomotion andpost-DCS analysis) or 60% (non-depleted/depleted analysis) of thepost-treatment bins were beyond the confidence interval limits. Thefiring rates depicted in the figures were smoothed with a moving averagewindow of 2 seconds. Spectrograms of LFPs were based on a 4 s window (1s sliding step) using multitaper method with five tapers (Percival etal, Spectral analysis for physical applications: multitaper andconventional univariate techniques (Cambridge University Press,Cambridge; New York, N.Y., USA, pp. xxvii, p. 583 (1993)), unlessotherwise stated.

For detecting changes in spectral index around spontaneous locomotion,the spectral index was calculated from a LFP spectrogram of 1.024 swindow sliding every 0.05 s in the periods around locomotion onset [−12s, 12 s] and in non-locomotion periods [−22 s, −12 s]. Locomotion periodspectral index values above a 99% confidence interval, based on aPoisson distribution from the no-locomotion spectral index values, wereconsidered significantly different.

Spike-LFP Entrainment Analysis

Units were classified as entrained to LFP when they displayed maximumSTA amplitude greater then 99.9% of 1000 simulated STAs with originalLFP segment and pseudo-random spike time stamps preserving the averagefiring rate. Preferred power of the STA was determined as frequenciesshowing power greater then 99% of the spectra of the simulated STA LFP.All STA LFPs were calculated in a [−256 ms+256 ms] window.

Statistics and Numerical Operations

For statistical tests, whenever distributions did not pass normalitytests (D'Agostino and Pearson omnibus normality test), non-parametricversions such as Mann-Whitney (t-test) or Kruskal-Wallis (ANOVA) wereused. In the latter case, Dunn's multiple comparison test was used as apost test if significant (p<0.05) differences were detected in theinitial test.

The variance for ratios were calculated as:

Var(X/Y)=E[Y ² ]Var(1/X)+Var(Y)E[1/X] ²

assuming E[X]=mean(X) and E[Y]=mean(Y). After that, Z-scores werecalculated for relevant comparisons of pairs of ratios as(mean(X)−mean(Y))/sqrt(Var(X)−Var(Y)) and Bonferroni corrected formultiple comparisons.

The number of bins used for histograms of locomotion speeds was adjustedaccording to the maximum speed scored in a single bin in the slowestcondition (i.e. 500 mm/s for the rats and 180 mm/s for the mice). Therelatively few (<0.3%) events with locomotion components faster than theupper limit found in the other conditions were assigned to the fastestbin. The number of events within each speed interval was summed and therelative change in response to DCS was calculated. A lower thresholdjust above the quantal step was used in the analysis of histogramdistributions, i.e. 10 mm/500 ms for both mouse and rat data.

Results

The potential of electrical stimulation of the dorsal column afferentpathways in the spinal cord was evaluated, as a strategy for treatingmotor symptoms in two animal models of PD. Experiments were aimed atestablishing both electrophysiological and behavioral effects of dorsalcolumn stimulation (DCS). This was achieved by chronically recording theneuronal activity from the dorsolateral striatum and primary motorcortex in animals placed in an open-field experimental set-up. Duringthese experiments, an investigation was made as to whether DCS, likeDBS, would allow for a reduction of L-DOPA doses used for treatment ofmotor symptoms.

Dopamine, Akinesia and Synchrony

The first set of experiments was carried out using an inducible mousemodel of PD, first in wild-type animals and then in dopamine-transporterknockout (DAT-KO) mice (Sotnikova et al, PLoS Biol. 3:e271 (2005)).Through pharmacological inhibition of dopamine synthesis, acute dopaminedepletion was induced in both types of animals in order to reliablydocument the effects of DCS during a severe parkinsonian-like state ofakinesia. In agreement with previous experiments, the characteristicbehavioral and electrophysiological changes following inhibition ofdopamine synthesis was confirmed in these animals (Carlsson, ActaNeurol. Scand. Suppl 51:11 (1972), Costa et al, Neuron. 52:359 (2006),Sotnikova et al, PLoS Biol. 3:e271 (2005)). In patients, the cardinalsymptoms of idiopathic PD, rigidity, resting tremor andbrady-/hypokinesia, have been reported to be clinically apparentfollowing degeneration of 60-70% of the dopaminergic neurons of thesubstantia nigra pars compacta, which in turn results in a 30-50%reduction of striatal dopamine levels (Brooks et al, Biol. Psychiatry59:908 (2006), Lloyd et al, J. Pharmacol. Exp. Ther. 195:453 (1975)). Inthese experiments, acute pharmacological dopamine depletion in wild-typeC57/BL6J mice was achieved with two i.p. injections (250 mg/kg) of thetyrosine hydroxylase inhibitor alpha-methyl-para-tyrosine (AMPT) duringa 6 h period. This treatment produced a 69% reduction of striataldopamine levels, as confirmed by dopamine tissue content analysis(mean±SD=4.5±2.0 ng dopamine per mg tissue in depleted animals comparedto 14.4±3.3 in controls; p<0.005 Mann-Whitney, n=6/6 (FIG. 11), slightlybelow the levels observed in PD patients (Brooks et al, Biol. Psychiatry59:908 (2006), Lloyd et al, J. Pharmacol. Exp. Ther. 195:453 (1975)).Equivalent symptoms to all the main clinical motor manifestations in PDpatients were found in AMPT-injected mice after the 6 h depletionperiod. Firstly, rigidity was displayed in the form of severe catalepsyassessed as the inability to remove the forepaws for at least 30 s froma bar placed 3 cm above the ground (under normal conditions animalsremove the forepaws immediately (Sotnikova et al, PLoS Biol. 3:e271(2005)). Secondly, a pronounced muscle tremor was evident during restingperiods, which was reflected in characteristic oscillatory patterns inelectromyographic (EMG) recordings obtained from chronically implantedmicrowires in the nuchal muscles (FIG. 12). This tremor was suppressedduring locomotion bouts. Thirdly, and perhaps the most evidentbehavioral change associated with the dopamine-depleted condition, therewas a substantial decrease in the amount of locomotion displayed in theopen-field. Dopamine-depleted animals showed a decrease in the averageamount of locomotion during testing periods that corresponded to −10% ofcontrol values [average locomotion scores in non-depleted and depletedanimals were (mean±SEM) 3.7±0.1 and 0.4±0.02 mm/s, n=11 and 14,respectively] (FIG. 13A, see also Drouot et al, Neuron 44:769 (2004) andSakai et al, Brain Res. 633:144 (1994)). Further, a detailed analysis oflocomotion episodes in terms of movement speed showed that the reductionwas most prominent for high- and medium-speed components reflecting anoticeable bradykinesia in the depleted state (locomotion scores indepleted animals expressed as percentage of the scores in non-depletedanimals were, slow: 10.5%, medium: 2.0%, fast: 0.3%; FIG. 13B).

In parallel with the overt changes in locomotive ability after dopaminedepletion, neuronal activity patterns of dorsolateral striatum andprimary motor cortex (MI) were also significantly altered. Differenceswere found both on a population level, through inspection of local fieldpotentials (LFPs), and in the firing patterns of single cortical andstriatal neurons. These were in agreement with previous studiesindicating prominent and rapid changes in corticostriatal neuronalensemble coordination after acute dopamine depletion (Costa et al,Neuron. 52:359 (2006)). To begin with, local field potentials (LFPs)were analyzed; these voltage fluctuations are considered to reflectalterations in the synaptic drive of neurons in the recorded area and,to some extent, the postsynaptic activation of populations of neuronssurrounding the recording site (Mitzdorf, Physiol. Rev. 65:37 (1985),Berke et al, Neuron. 43:883 (2004)). An example of LFP spectrogramsrecorded in MI during two 5-min periods before and after dopaminedepletion is shown in FIG. 13C (second and third rows, left and right,respectively). The spectral shift to lower frequencies in the depletedstate is evident. Spectral analysis revealed particularly powerfuloscillations around 1.5-4 Hz and in the lower beta range (10-15 Hz),whereas the power of oscillations >25 Hz was decreased in relation tobaseline conditions (standardized spectrograms, FIG. 13C, third row andFIG. 14). Note, however, that the spectral power was somewhat normalizedduring the brief locomotion episode at the end of the 5-minute recordingperiod.

In general, high-frequency oscillations were more prominent in thenon-depleted relative to the depleted condition. Still, low-frequencyoscillations could be found in non-depleted animals during restingperiods, although these oscillations were typically not in the samefrequency range as those found during dopamine depletion, but rather athigher band around 4-9 Hz. When analysing single- and multi-unitactivity in the normal and dopamine-depleted states, importantdifferences were also found. The firing rates of a majority of 52striatal and cortical neurons, which were positively identified after 6hour depletion period, showed significant differences (70.0% in motorcortex and 75.0% in striatum, α=0.001) when the more active non-depletedstate and the immobile depleted condition were compared (as exemplifiedby the activity raster plots shown for a few units in FIG. 13C, bottomrow). It was also observed that during dopamine depletion, a higherproportion of neurons tended to discharge phase-locked to LFPoscillation, in effect resulting in increased synchronicity (overall,52.7% [64/129] of the recorded units showed entrainment of actionpotentials to the LFP oscillations after dopamine depletion as comparedto 37.0% [44/127] in the non-depleted condition; α=0.001; FIG. 14).

Taken together, the robust behavioral changes and the characteristicdifferences found between the depleted and non-depleted states withregard to both LFP oscillations and firing patterns of populations ofsingle neurons confirmed that acute dopamine depletion provided areliable experimental model for comprehensively characterizing theeffects of DCS specific to the Parkinsonian state.

DCS Alleviates Akinesia and Synchrony

The effect of DCS was next evaluated in mice before and after acutepharmacological dopamine depletion. DCS was achieved by chronicimplantation of custom-made flat bipolar platinum electrodes positionedepidurally above the dorsal columns of the spinal cord at the upperthoracic level (FIGS. 13E and 13F). Animals were stimulated for 30 severy tenth minute. Four DCS paradigms were used: continuous stimulationat 10, 100 or 300 Hz, or stimulation in trains of 8 pulses at 100 Hzevery two seconds. In addition to electrical stimulation, air-puffs(0.25 Hz targeted directly to the animal) either alone or in combinationwith 100 Hz continuous DCS were also tested, completing a 1-h cycle ofsix stimulation paradigms. The same stimulation cycle was repeated threetimes, both before and after dopamine depletion (FIG. 15). Finally, in aseparate set of experiments, the effect of bilateral trigeminal nerve(infraorbital branch) stimulation was investigated using the sameexperimental paradigm (rightmost bars in FIG. 16A). Stimulationintensities were individually adjusted according to the responsethresholds of each animal and each stimulus type. Typically, a startleresponse or a sudden change in whisking behaviour was taken to indicatethreshold level (T) for response to stimulation. For DCS ˜1.3 T was usedin all experiments (mean±SD at 300 Hz=333±138 μA), while for trigeminalnerve stimulation ˜1.1 T was used (mean±SD at 300 Hz=54±20 μA) in orderto avoid activating nociceptive afferent fibres present in this nerve.

After three stimulation cycles under control (non-depleted) conditions,animals were injected with AMPT and acquisition of data related to thedopamine-depleted state commenced when animals displayed clear catalepsy(according to the test criterion described above). It was found that DCShad a dramatic effect on the amount of locomotion displayed duringstimulation periods in the dopamine-depleted animals. This effect wasstrongest for 300 Hz stimulation; on average the amount of locomotionduring stimulation periods was more than 26 times higher than during the5 minute period prior to stimulation (FIG. 16A). DCS had a smaller,albeit clear effect, using lower stimulation frequencies (FIG. 16A andFIG. 17). In contrast, air-puffs alone or trigeminal nerve stimulationwere not effective, suggesting that the locomotive recovery observed isspecific to the stimulation of dorsal column pathways and is not a mereconsequence of arousal or pain. DCS caused increased locomotion alsoduring non-depleted conditions, but this increase was moderate (4.9times pre-stimulus values at 300 Hz) in comparison to that in depletedanimals (26 times; FIG. 16A). Locomotion was normally initiated a fewseconds after the onset of DCS, with a slightly longer delay in depletedanimals (median=3.35/1.35 s, interquartile range=2.22/1.22 s, p=0.023,Mann-Whitey, in depleted/non-depleted animals, for 300 Hz stimulation).However, a small residual effect was also found after high-frequencystimulation in depleted, but not in non-depleted animals (3.4 and 0.95times pre-stimulus values, respectively for 300 Hz DCS during the 30 sfollowing stimulation). In addition to the strong improvement in generallocomotive capability, DCS also proved efficient for alleviation ofbradykinesia as indicated by the relatively larger increase in theamount of fast movement components in depleted animals (FIG. 16B).

Struck by the dramatic functional recovery on the behavioral level, theelectrophysiological changes associated with motor improvements wereanalyzed next. Analysis of LFP recordings during DCS in both MI and instriatum showed a strong shift in spectral power from lower frequenciesto higher (average spectrograms from a total of 21 events of DCS at 300Hz obtained from 9 animals are shown in FIG. 16C). The spectral shiftwas maintained throughout the stimulation period and lasted for ˜50 sfollowing the off-set of stimulation. This indicates a prolonged shiftin the activity state of these brain structures, also ruling out anymajor effects of contamination in the spectrograms from stimulationcurrents (FIG. 16C). To condense the spectral shift into a singlemeasure, a spectral index was computed by dividing the spectral rangeanalyzed into an upper and lower half and calculating the ratio of thesummed power of the frequencies in the two intervals [(25-55 Hz)/(1.5-25Hz)]. The spectral index (black trace in FIG. 16C) clearly illustratesthe rapid spectral shift induced by DCS and the prolonged effect afterDCS had ceased.

DCS also affected the firing patterns of individual neurons. To avoidinterference from stimulation artefacts, the 30 second stimulationperiods were excluded from the analysis of spike data. But even duringthe period following stimulation, numerous neurons showed significantlyaltered firing rates (47.9% in MI and 41.8% in striatum, α=0.01; FIG.16C, row 4 and 5, respectively). The fraction of units entrained to LFPdropped notably (from 42.7/38.8% in MI/striatum the 30 s before DCS to24.5/24.0% the 30 s after DCS, α=0.01). Thus, DCS was shown to have aprominent effect on activity patterns in both MI and in striatum and toreduce the aberrant synchronization representative of the dopaminedepleted state.

Although the onset of locomotion was delayed a few seconds, changes inthe neural activity were detected almost immediately following DCS onset(mean±SD evoked potential latency=44±5 ms), perhaps indicating that theelectrophysiological changes have a permissive rather than directlyinstructive role for the initiation of locomotion. It is consequently anintriguing possibility that the impressive behavioral improvementsobtained with DCS are mediated by desynchronization of the activity ofcorticostriatal circuits, an event that may create a state permissive ofmovement initiation, even in Parkinsonian animals, where initiation ofmovement is otherwise impaired.

A Brain State Permissive of Locomotion

In the initial characterization of the dopamine-depleted state above itwas noted that during the relatively rare instances when the depletedanimals displayed locomotion, low-frequency oscillations were diminished(FIG. 16C). This situation bears an obvious resemblance to the DCSinduced state. Thus, a certain decrease of low-frequency oscillationsmay be required to initiate locomotion. To get a better understanding ofthe changes in the activity of MI and striatum neurons associated withthe initiation of locomotion, the detailed temporal patterns of shiftsin oscillatory LFP activity were analyzed during spontaneous locomotionevents in non-depleted (115 events in 10 animals) and depleted mice (51events in 5 animals) (FIGS. 18A and 18B). In both states, significantspectral shifts (p<0.01, see Experimental Details) from lower to higherfrequencies were detected a number of seconds prior to the initiation oflocomotion (non-depleted: mean±SD=2.9±1.7 s, range 0.1-5.5 s, n=88, MIand striatal LFPs; depleted: 3.0±1.7, range 0.2-5.5 s, n=48, MI andstriatal LFPs). Yet, there were also important differences, most notablybelow 25 Hz. A more differentiated decrease in power of oscillationsbelow 8 Hz and an increase above 17 Hz was observed in non-depletedanimals, whereas the spectral power in a broader range between 5 and 25Hz was decreased in depleted animals. Since these different patternsoccurred before the onset of locomotion, it is unlikely that they weredue to differences in locomotion between the two groups. Instead, theycould be part of the explanation why depleted animals moved slower andfor shorter time periods. In addition to LFP activity patterns, strikingparallels between spontaneous locomotion and DCS-induced brain changesalso existed on the level of single neurons. In fact, the same type offiring rate changes that were found following DCS also occurred inconjunction with spontaneous locomotion events. From a total pool of 193neurons (from 9 control and 5 dopamine-depleted recordings sessions in11 animals), 111 modulated their firing rate during locomotion andunexpectedly, 59 of these neurons showed a pattern of early activation,2.9±1.4 s (mean±SD) before actual locomotion onset (range=0.5-4.5 s,n=59 striatal and MI units from depleted and non-depleted conditions,FIG. 18C).

Altogether, the close electrophysiological parallels between DCS andspontaneous locomotion suggest that DCS brings the activity ofcorticostriatal circuits in dopamine-depleted animals back to a brainstate that normally precedes locomotion. Accordingly, DCS could in thisway facilitate initiation of locomotion in the Parkinsonian state.

DCS in Combination with L-DOPA Treatment

In spite of improved techniques for DBS, pharmacological treatment stillremains the preferred choice for most PD patients. However, an importantfeature of DBS is that it permits a reduction of L-DOPA dosage, therebyminimizing the long-term side effects associated with pharmacotherapy(Perlmutter et al, Annu. Rev. Neurosci. 29:229 (2006)) and extending thebeneficial time period of pharmaceutical treatment. Because theimplantation of an epidural stimulation electrode over the dorsalcolumns constitutes a substantially less invasive surgical procedurethan the implantation of DBS electrodes, a combined effect of DCS withlower doses of L-DOPA could potentially allow more patients to benefitearly on from this combined therapy, while improving their quality oflife. Therefore, the extent to which DCS could substitute for, orreduce, the L-DOPA doses needed to allow for locomotion was evaluated.

In order to find the minimum dose of L-DOPA (alone or combined with DCS)required to restore locomotion, a severely dopamine-depleted akineticanimal model was used, namely mice lacking the gene for the presynapticdopamine membrane transporter (DAT-KO). Since the transporter normallyrecycles a large portion of the released dopamine, these micedisplay >95% decrease in the striatal content of dopamine (Sotnikova etal, PLoS Biol. 3:e271 (2005)). These levels of dopamine can be furtherdecreased to virtually undetectable levels by a pharmacogeneticapproach, injecting AMPT (250 mg/kg i.p.) to inhibit the synthesis ofdopamine, resulting in a completely akinetic animal model (Sotnikova etal, PLoS Biol. 3:e271 (2005)). Thus, by gradually increasing dopaminelevels through repeated L-DOPA injections every hour, it was possible toprobe the locomotion thresholds. DCS (1.3 T at 300 Hz) was applied everyfifth minute, from 15 to 35 minutes post-injection in the L-DOPA+DCSgroup (it was during this time period that animals were observed tostart moving in the L-DOPA only group). In the group receiving onlyL-DOPA injections (n=6 sessions from 4 mice), locomotion typically firstoccurred after the fifth injection (5 mg/kg dose, corresponding to atotal dose of 15 mg during the first five hours). When L-DOPA treatmentwas combined with DCS, the same amount of locomotion was displayed afterthe second injection (2 mg/kg dose, corresponding to a total dose of 3mg in the first two hours) (n=10 experiments from 7 mice, FIG. 19A).That means that ⅕ of the L-DOPA total dose, when used in combinationwith DCS, was enough to produce equivalent locomotion effects achievedwhen L-DOPA alone was chosen as a treatment. In fact, overall, there wasalso a general increase in the amount of locomotion displayed in theL-DOPA+DCS group over the entire range studied. Thus, L-DOPA+DCS seemsto be superior to L-DOPA alone in terms of the ability to rescuelocomotive capability after severe dopamine depletion. Finally, it wasnoted that animals in the L-DOPA+DCS group consistently showed highervalues of spectral index than the L-DOPA only group. This suggests thatDCS facilitates locomotion, even in severely depleted animals, throughsimilar mechanisms (FIG. 20).

DCS is Effective after Chronic Lesions

Although the acute dopamine depletion model employed in the first set ofexperiments was shown to reproduce all the main symptoms of PD, it wasimportant to confirm the effectiveness of DCS in an animal model thatalso involves loss of nigrostriatal dopaminergic connections. In theseexperiments, chronic dopaminergic denervation of the striatum wasachieved using 6-OHDA lesions in rats (n=4). After bilateral 6-OHDAlesions (three sites per side), rats displayed progressive deteriorationof motor function and sustained weight loss, both cardinal signs ofsuccessful lesioning in this widely used animal model (Cenci et al, Nat.Rev. Neurosci. 3:574 (2002), Winkler et al, Neurobiol. Dis. 10:165(2002)). One month after lesioning, several motor impairments wereapparent, including abnormal posture and gait, hypokinesia,bradykinesia, and reduced forelimb dexterity (for example manipulatingfood pellets). When placed in the open-field, 6-OHDA lesioned ratsdisplayed reduced locomotion compared to another group of rats (n=4),which received vehicle injections in identical sites in the striatum(mean±SEM=2.85±0.068 and 7.78±0.144 mm/s on average, respectively).Quantification of immunohistochemical staining of the dopaminesynthesizing enzyme tyrosine hydroxylase in brain sections ofsham-lesioned and 6-OHDA treated rats indicated that lesioned rats hadonly ˜20% of the immune-signal found in sham-lesioned animals (FIG. 21),confirming a specific effect of 6-OHDA on the loss of dopaminergicneurons.

Lesioned rats were then tested during two one-hour sessions in theopen-field, the first hour without stimulation and the second with DCSapplied for 30 s every tenth minute (1.3 T, 300 Hz, mean±SD intensityfor sham and lesioned: 286.6±119.9 μA and 233.4±53.7 μA, respectively).In the lesioned group, DCS resulted in remarkably increased amounts oflocomotion compared to the first hour, whereas sham animals actuallymoved less during DCS sessions than during the non-DCS sessions (FIG.19C). Hence, in agreement with the findings in the wild-typedopamine-depleted mouse model (FIG. 16A), there were also specificimprovements of motor function in the Parkinsonian state compared tocontrols in a chronic lesion model of PD. In lesioned rats, DCS not onlyalleviated hypokinesia during stimulation, but it also caused anincrease in locomotion after the stimulation period. This residualeffect lasted around 100 s. (FIG. 19B). Comparing the average distancetraveled per second in DCS and non-DCS sessions for lesioned and shamanimals, respectively, yielded significant differences between all fourconditions (p<0.001, Kruskal-Wallis and Dunn's multiple comparison test,FIG. 19C).

The effect of DCS on bradykinesia in 6-OHDA lesioned rats was alsoevaluated. The relative change in the number of locomotion events scoredin each movement speed interval (slow, medium and fast) for DCS comparedto non-DCS sessions was calculated for sham treated and lesionedanimals. Lesioned animals showed a relative increase in the number ofscored locomotion events for all movement speeds. This effect was morepronounced for faster movements, indicating a specific effect onbradykinetic symptoms in addition to the general improvement in theoverall amount of locomotion (FIG. 19D). In summary, a similarrestoration of locomotive capability, which was seen in the first set ofexperiments using acute dopamine depletion, was also observed inchronically lesioned animals. This further confirmed the potential ofDCS as a therapeutic approach for PD, following extensive nigrostriataldenervation.

In summary, the studies described above demonstrate that stimulation ofthe dorsal column pathways using epidural implanted bipolar electrodes,a simple, easy to perform, semi-invasive method, can restore locomotivecapability in two animal models of PD symptoms: acutelydopamine-depleted mice and rats with dopaminergic neuronal loss. Inparallel with the dramatic behavioral improvements, DCS was found toshift activity patterns in the primary motor cortex and in thedorsolateral striatum into a state closely resembling that found priorto and during initiation of locomotion in normal and depleted animals.Based on these findings, it is proposed that DCS helps motor relatedbrain areas shift into a permissive state for the initiation ofmovements, in part by effectively desynchronizing cortical and striatalactivity patterns. This notion is also supported by the fact that bothspontaneous locomotion and locomotion triggered by DCS displayed thesame electrophysiological characteristics and similar latency in theonset of locomotion in relation to the preceding shift in neuronalactivity patterns. The effect of DCS on motor related brain areasrecorded in the present study may involve activation of brainstemarousal systems (Fanselow et al, J. Neurosci. 20:8160 (2000)) and/ordirect activation of the involved neuronal circuits through thelemniscal/thalamic pathways. Still, as neither air-puffs alone norstimulation of trigeminal nerve afferents induced locomotion, eventhough both stimuli clearly caused an arousal response in the animals,it is unlikely that activation of arousal systems alone can explain theeffect of DCS.

The electrophysiological data suggest possible mechanisms for thesuccess of DCS in the treatment of PD, based on existing theories ofbasal ganglia pathology in PD and specifically considering the circuitryknown to be involved in initiating voluntary locomotion (FIG. 22). Thecommand to the spinal cord to initiate locomotion, via reticulospinalpathways, is issued by the diencephalic and mesencephalic locomotorregions. However, a prerequisite for these midbrain structures to becomeactive and trigger locomotion is that they are disinhibited by theoutput nuclei of the basal ganglia, which in turn need to bedisinhibited by striatum (Grillner et al, Trends Neurosci. 28:364(2005)). Under normal circumstances, the cortex has a powerfulexcitatory influence on the striatum. In contrast, with reduced striataldopamine levels, the activation threshold of the projection neurons fromthe striatum is significantly increased (Grillner et al, Brain Res. Rev.57:2 (2008)), making it less likely that cortical input to the striatumwill be conveyed through this pathway disinhibiting basal ganglia outputnuclei. As a consequence, brainstem motor regions remain under tonicinhibition, and the initiation of goal directed locomotion and othertypes of volitional motor activity become impaired. In this context, DCSmay exert its effect by activating large cortical areas, increasing thecortical and thalamic input to the striatum. This may in turn, promotethe depolarization and, consequently, facilitate the activation ofstriatal projection neurons. Another important consequence of thereduced cortical control of striatum at low dopamine levels is that boththalamic and internally driven striatal low-frequency oscillationsbecome more prominent in this situation (Smith et al, Trends Neurosci.27:520 (2004), Wilson, Neuron 45:575 (2005)). These oscillations maylead to increased synchronicity because the generation of actionpotentials tends to occur at distinct phases of the LFP oscillation(Costa et al, Neuron. 52:359 (2006), Berke et al, Neuron 43:883 (2004)).This was confirmed in the experiments in which both motor cortex andstriatum showed excessive low-frequency synchronized oscillatoryactivity in dopamine-depleted animals and an increased entrainment ofspikes to low-frequency components of the LFPs. Accumulating evidenceindicates that such synchronous activity interferes with normalinformation processing in these circuits and should be consideredpathogenic in PD (Hammond et al, Trends Neurosci. 30:357 (2007)). In thedata presented here, it is shown that DCS effectively abolishes aberrantsynchronous low-frequency oscillations. It is, therefore, tempting tospeculate that the suppression of low-frequency oscillations isparticularly important for amelioration of motor symptoms in PD. Thenotion that a certain suppression of low-frequency oscillatory activityis necessary for the initiation of voluntary movements also has somesupport from previous studies investigating basal ganglia activityduring other types of volitional movements (Kuhn et al, Brain 127:735(2004), Courtemanche et al, J. Neurosci. 23:11741 (2003), Amirnovin etal, J. Neurosci. 24:11302 (2004)).

Finally, a particularly important finding in this study was thedemonstration that the combined effect of L-DOPA and DCS allowed forrecovery of motor function at significantly lower doses of L-DOPA inseverely dopamine-depleted animals. The considerably less invasivenature of the epidural DCS electrode compared to DBS electrodessuggests, therefore, that DCS could be a particularly attractivecomplement for treatment of symptoms of PD in earlier stages of thedisease. In this way, the use of DCS in combination with low levels ofL-DOPA could potentially be of great help to large numbers of patients,beginning in the early stages of diagnosis.

All documents and other information sources cited above are herebyincorporated in their entirety by reference.

1-17. (canceled)
 18. A method of continuous therapy for disruptingpathological synchronous neural activity in the brain of a patientsuffering from a psychiatric disorder, the method comprising:continuously stimulating a peripheral cranial nerve, dorsal sensoryroots of spinal nerves, or posterior funiculi of the spinal cord of saidpatient, wherein the continuous stimulation consists of tonic electricalstimulation under conditions such that said neural activity isdisrupted.
 19. The method according to claim 18, wherein saidpsychiatric disorder is epilepsy, depression, or obsessive compulsivedisorder.
 20. The method according to claim 18, wherein said electricalstimulation is delivered by an implanted peripheral nerve cuffelectrode.
 21. The method according to claim 18, wherein saidstimulation disrupts pathological synchronous activity in the motorcortex or basal ganglia of said patient.
 22. The method according toclaim 18, wherein an infraorbital nerve is stimulated.
 23. The methodaccording to claim 18, wherein posterior funiculi are stimulated. 24.The method according to claim 23, wherein said stimulation is deliveredwith electrodes positioned epidurally above the dorsal column of saidpatient.
 25. The method according to claim 18, wherein a trigeminalnerve is stimulated.
 26. The method according to claim 25, wherein saidtrigeminal nerve is stimulated transcutaneously.
 27. The method of claim18, wherein said stimulation comprises biphasic square constant currentpulses.
 28. The method of claim 27 wherein said pulses are emitted at afrequency of 100 to 300 Hz.
 29. The method of claim 28 wherein saidbiphasic pulses have pulse durations between 250 us and 1 msec.
 30. Themethod of claim 18 comprising continuously stimulating posteriorfuniculi of the spinal cord of said patient.
 31. A method of continuoustherapy for disrupting pathological synchronous neural activity in thebrain of a patient having epilepsy, depression, or obsessive compulsivedisorder, said method comprising: continuously stimulating dorsalsensory roots of spinal nerves or posterior funiculi of the spinal cordof said patient using tonic electrical stimulation via epiduralimplanted bipolar electrodes under conditions such that said neuralactivity is disrupted, wherein said method does not include acquiringfield potential data indicative of a the patient's electrical brainactivity in real-time and analyzing the field potential data to identifyseizure-related brain activity.
 32. The method according to claim 31,wherein said patient is receiving dopamine.
 33. The method according toclaim 32, wherein said stimulation modulates corticostriatal activity.